Darkfield illumination is most readily set up at low magnifications even up to 100x, even though it can be used with any dry objective lens. Any time you wish to view everything in a liquid sample, debris and all, dark field is best. In darkfield microscopy, to view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye. Instead of coming up through the specimen, the light is reflected by particles on the slide. Everything is visible regardless of color, usually bright white against a dark background. Pigmented objects are often seen in false colors, that is, the reflected light is of a color different than the color of the object. Better resolution can be obtained using dark as opposed to bright field viewing. Sophisticated equipment is not necessary to get a dark field effect, but you do need a higher intensity light, since you are seeing only reflected light. At low magnification, up to 100x, any decent optical instrument can be set up so that light is reflected toward the viewer rather than passing through the object directly toward the viewer. To set up a dissecting microscope however, for dark field viewing, the specimen should be placed over an opening so that light reflects only from surfaces between cover slip and slide, not from a surface beneath the slide. You may need to make a stand to hold the slide. The surface beneath the opening should be a flat black. Turn off any built-in illuminator. Aim a high-intensity light source toward the specimen at an angle, from the top or side through a glass dish or jar. Using a high powered microscope, with a compound microscope, dark field is obtained by placing an occulting disk in the light path between source and condenser.
A cheap set of occulting disks can be prepared by cutting circular pieces of black electrical tape ranging from about the size of a five pence piece up to a diameter that equals the width of the slide, and sticking them to the slide in a row. The circles should be spaced well apart. A specimen is placed on the microscope stage as usual, and the illumination should be made as uniform as possible. If there is an aperture diaphragm in the condenser, it should be opened up wide. After focusing at low power, the slide with occulting disks is placed in the light path between source and condenser, bringing it as close to the bottom of the condenser as it will go. Begin with the largest disk, sliding it around until it is directly in the center of the light path. Increasing the illumination should then produce a good dark field effect. To optimize, first try stopping down the field diaphragm to get the best contrast between background and specimen. Match the size of the occulting disk to the field diameter, so that the edge of the disk is just outside the field of view - smaller disks are appropriate for higher power objectives. Vertically, the disk should be a close to the condenser as possible, to make the contrast the greatest. On microscopes with built-in dark field equipment, the view is so impressive because the occulting disk is built into the condenser - very close and focused. After testing the set-up this way, a stand might be rigged to fit under the microscope, so the slide can be placed in position without holding it. Something that grabs the condenser and supports the occulting disks would be ideal. The less you have to mess with, the better. While specimens may look washed out and lack detail in bright field, protists, metazoans, cell suspensions, algae, and other microscopic organisms are clearly distinguished and their details show up well. Suspensions of cells and samples of pond water look spectacular in dark field. At 100x you can readily see bacteria, even distinguish some structure like rods, curved rods, spirals, or cocci, and movement. Non-motile bacteria look like vibrating bright dots against a dark background. Motile bacteria can be seen moving in a definite direction, sometimes remarkably fast. Even tiny dust particles are obvious. Darkfield is especially useful for finding cells in suspension. Darkfield makes it easy to find the correct focal plane at low magnification for small, low contrast specimens.
